Toxicology in Vitro 36 (2016) 180185

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Toxicology in Vitro

journal homepage: www.elsevier.com/locate/toxinvit

Toxicity analysis of ocular prosthesis acrylic resin with or without

pigment incorporation in human conjunctival cell line
Emily Vivianne Freitas da Silva a, Marcelo Coelho Goiato a,, Liliane da Rocha Bonatto a,
Rodrigo Antonio de Medeiros a, Daniela Micheline dos Santos a,
Elidiane Cipriano Rangel b, Sandra Helena Penha de Oliveira c
a
Department of Dental Materials and Prosthodontics, Aracatuba Dental School, Sao Paulo State University (UNESP), Jose Bonifacio St., 1153, Vila Mendonca, Aracatuba, Sao Paulo, Brazil
b
Department of Basic Sciences, Aracatuba Dental School, Sao Paulo State University (UNESP), Jose Bonifacio St., 1153, Vila Mendonca, Aracatuba, Sao Paulo, Brazil
c
Technological Plasma Laboratory (LaPTec), Experimental Campus of Sorocaba, UNESP, 3 de Marco Av., Alto da Boa Vista, Sorocaba, Sao Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to evaluate the inuence of pigment incorporation on the cytotoxicity of ocular pros-
Received 14 April 2016 thesis N1 color acrylic resin. Nine samples were manufactured by heat-polymerization in water bath and divided
Received in revised form 5 August 2016 into 3 groups: acrylic resin without pigment incorporation (group R), acrylic resin with pigment incorporation
Accepted 8 August 2016
(group RP), and acrylic pigment (group P). Eluates formed after 72 h of sample immersion in medium were in-
Available online 10 August 2016
cubated with conjunctival cell line (Chang conjunctival cells) for 72 h. The negative control group consisted in
Keywords:
medium without samples (group C). The cytotoxic effect from the eluates was evaluated using MTT assay (cell
Dental material proliferation), ELISA assay (quantication of IL1, IL6, TNF and CCL3/MIP1) and RT-PCR assay (mRNA expres-
Ocular prosthesis sion of COL IV, TGF and MMP9). Data were submitted to ANOVA with Bonferroni post-tests (p b 0.05). All
Acrylic resins groups were considered non-cytotoxic based on cell proliferation. However, resin with pigment incorporation
Biocompatibility testing showed signicant IL6 quantity increase. Resin without pigment incorporation exhibited higher mRNA expres-
Monomers sion of COL IV, MMP9 and TGF , however it was also observed for the negative control group. The materials ex-
hibited divergent biological behavior. Despite the pigment incorporation that resulted in an increase of IL6, no
cytotoxicity was observed based on cell proliferation.
2016 Elsevier Ltd. All rights reserved.

1. Introduction polymerization reaction that consists of the conversion of monomers

The ocular prosthesis is commonly used in anophthalmic patients
and is generally made of N1 acrylic resin, which mimics the patient
sclera, and colorless acrylic resin, responsible for the prosthesis charac-
terization covering. The acrylic resin is an option of choice due to its
handling and adjustment, satisfactory aesthetics and low cost (Goiato
et al., 2012, 2014; Andreotti et al., 2014; Fernandes et al., 2009a).
Acrylic pigments with different colors can be incorporated into
acrylic resin for articial sclera, aiming to simulate the natural sclera
of the patient. Moreover, the articial iris is positioned and red silk bers
may also be incorporated to mimic blood vessels. Then, a thin layer of a
colorless resin is used to cover the articial iris and silk bers, providing
a natural appearance to the prosthesis (Goiato et al., 2010a, 2014).
When the liquid, which consists of a methyl methacrylate (MMA)
monomer, is mixed with the powder, an MMA polymer, there is a
Corresponding author at: Department of Dental Materials and Prosthodontics,
Aracatuba Dental School, Sao Paulo State University - UNESP, Jose Bonifacio St., 1153,
Vila Mendonca, Aracatuba, Sao Paulo 16015-05, Brazil.
E-mail address: goiato@foa.unesp.br (M.C. Goiato).
into polymers, optimizing the physical properties of the material
(Bural et al., 2011a). If this reaction is incomplete, residual MMA mono-
mers and other chemicals that can be toxic can be released. Examples of
such products are benzoic acid, formaldehyde, and methacrylic acid,
among others (Bural et al., 2011a,b; Chaves et al., 2012; Tay et al.,
2012; Att et al., 2009; Fernandes et al., 2009b).
Aesthetics, durability and proper adaptation are some of the essen-
tial requirements of the ocular prosthesis. However, biocompatibility
is a critical characteristic for the success of the treatment and can be
evaluated through in vitro cytotoxicity tests, such as the method of cell
cultures, which has relatively simple performance and reproduction
conditions (Goiato et al., 2010a; Fernandes et al., 2009b; Borra et al.,
2009; Jorge et al., 2007; Saravi et al., 2012).
Preferably, primary cells or cell lines, which are closest to the target
organ, should be used (Jorge et al., 2007), the use of the cell line from
human conjunctiva (Wong Kilbourne derivative of Chang conjunctiva)
has been widely reported in in vitro studies of ophthalmic products
(Clouzeau et al., 2012; Ayaki et al., 2011a,b, 2012). Therefore, this cell
line can be used for the assessment of the cytotoxicity of materials
used for ocular prosthesis confection, since its support tissue is the

http://dx.doi.org/10.1016/j.tiv.2016.08.005
0887-2333/ 2016 Elsevier Ltd. All rights reserved.
 E.V.F. da Silva et al. / Toxicology in Vitro 36 (2016) 180185
conjunctiva, a thin membrane responsible for eye protection
(Barisani-Asenbauer et al., 2013; Willoughby et al., 2010).
The biocompatibility of the ocular prosthesis was not previously
evaluated, however the necessity to know this property is real and
important to guarantee its secure clinical use in patients. Therefore,
this study aimed to evaluate the inuence of pigment incorporation
on the cytotoxicity of ocular prosthesis N1 color acrylic resin.
This evaluation was performed through the analysis of the cell
proliferation by MTT assay, and the production of proinammatory
cytokines and extracellular matrix proteins by a human conjunctival
cell line.
The null hypothesis is that the N1 color acrylic resin, with or without
acrylic pigment, and the isolated acrylic pigment do not produce toxic
effects on the cell line studied.
2. Materials and methods
The number of samples was determined based on previous stud-
ies (Bural et al., 2011a; Jorge et al., 2004; Retamoso et al., 2014), and
based on the results of a pilot study. A power analysis was performed
to determine the number of specimens required for the study,
aiming to provide sufcient power (over 95%). Therefore, 3 samples
were used.
Nine samples of materials used for making ocular prosthesis were
manufactured by heat-polymerization in water bath (Table 1). These
samples were divided into 3 groups (Saravi et al., 2012): acrylic resin
without pigment incorporation (R, resin), acrylic resin with pigment
incorporation (RP, resin with pigment), and acrylic pigment
(P, pigment).
The samples were 10 mm in diameter and 3 mm in thickness
(Monteiro et al., 2012) and were manufactured through auto-polymer-
ized resin samples, previously obtained from a metallic matrix, which
were included in asks (Artigos Odontolgicos Clssico Ltda, Sao
Paulo, Brazil) by using type IV dental stone (Durone, Dentsply Ind e
Com Ltda, Rio de Janeiro, Brazil) and extra-hard laboratory silicon
(Zetalabor, Zhermack, Rovigo, Italy), for embedding the molds. Then,
the asks were opened and the molds were obtained after the samples'
removal (Goiato et al., 2010a,b).
The acrylic resin (R) and the isolated acrylic pigment (P) were pro-
portioned and mixed according to the manufacturer's instruction and
positioned into the molds contained in the asks. For the RP group,
the incorporation of acrylic pigment was performed during the resin
mixing. For this, the acrylic resin and pigment were properly weighed
on a precision digital scale (BEL Equipamentos Analtico, Sao Paulo, Bra-
zil) and the pigment was equivalent to 7% of the acrylic resin weight
(Goiato et al., 2013).
After the materials' insertion in the molds contained in the asks, a
counter-ask was positioned and raised in a hydraulic bench press
with a 1.250 kgf weight for 2 min (Goiato et al., 2012). Posteriorly, poly-
merization was executed according to the manufacturer's instructions,
initiated with bench polymerization, immersion of the ask in water
Table 1
Material, commercial brand and chemical composition of the groups.
Material Commercial brand Chemical composition
Acrylic N1 acrylic resin (Artigos MMA polymer,
resin Odontolgicos Clssico Ltda, Sao dibuthylftalato, ethyl acrylate,
Powder Paulo, Brazil) pigments
Acrylic Poli-Cr (color R2) (Artigos MMA polymer,
pigment Odontolgicos Clssico Ltda, Sao dibuthylftalato, ethyl acrylate,
Powder Paulo, Brazil) around 1.5% of various organic
and inorganic pigments
Acrylic Clssico (Artigos Odontolgicos MMA monomer, topanol
resin Clssico Ltda, Sao Paulo, Brazil)
Liquid
181
(room temperature), mild heating for 30 min, no heating for 30 min
and boiling for one hour. After these periods, the samples were removed
from the asks and a samples' nishing was performed with a Maxi-Cut
abrasive drill (Vicking, Sao Paulo, Brazil).
An additional specimen of each group was used for the analysis of
the surface chemical composition. This analysis was performed on
small volumes (on the order of 1 m3), through energy-dispersive spec-
troscopy (EDS).
For the cytotoxicity analysis, three samples from each group were
placed into a sterile vial with 10 mL of Medium 199 (Gibco, New York,
United States) supplemented with 10% fetal bovine serum (FBS) and in-
cubated at 37 C for 72 h. During this period, substances were leached
for the medium. Then, the eluates were ltered through 0.22 m lters
(Millex, Millipore, Darmstadt, Germany) for sterilization (Att et al.,
2009; Jorge et al., 2007; Saravi et al., 2012; International Organization
for Standardization [homepage], 2009).
To evaluate the possible toxic effect of substances released by the
groups, the cell culture of the human conjunctiva cell line (Wong
Kilbourne derivative of Chang conjunctival cell line, clone 1-5c-4) was
the selected method. This cell line was obtained from the American
Type Culture Collection (CCL-20.2, Virginia, United States). The cells
were expanded in asks with Medium 199. The medium was supple-
mented with 10% FBS, 10 g/mL penicillin, 10 g/mL streptomycin,
10 g/mL gentamicin and 250 g/mL fungizone. The cells were incubat-
ed with 5% CO2 and controlled humidity at 37 C (Clouzeau et al., 2012;
da Silva et al., 2016).
The cells were expanded until cell suspensions of 5 104 cells/mL
were achieved, predetermined by a pilot study. In a 24 well plate,
1 mL of this suspension was pipetted into each well and after 24 h of in-
cubation in a humidied atmosphere (5% CO2 and controlled humidity
at 37 C in an incubator), the medium was replaced by 500 L of eluates
from different groups. Negative control (group C, control) consisted in
culture medium with 10% FBS and without samples (Att et al., 2009;
Clouzeau et al., 2012). Medium with Tween 20 (Sigma-Aldrich, Missou-
ri, United States) served as a positive control (group T, Tween). The
plate was incubated for 72 h with the same incubation and temperature
conditions determined for generation of the eluates.
The culture medium was discarded and 500 L of Medium 199 with-
out FBS and with 0.5 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide) was pipetted into each well. The plate
was incubated in the same cited conditions for 4 h (Att et al., 2009;
Jorge et al., 2007; Gonalves et al., 2008). Later, the culture medium
was removed and the intracellular formazan was released by solubiliza-
tion with 1 mL of isopropanol per well. The plate was agitated for 5 min
and the absorbance (570 nm) was measured, in triplicate, using a UV
visible spectrophotometer (SpectraMax 190, Molecular Devices, Califor-
nia, United States) (Att et al., 2009; Saravi et al., 2012; Gonalves et al.,
2008).
For the Enzime-linked immune-absorbent assay (ELISA) (DuoSet
ELISA Development Systems, R&D System, Minnesota, United States),
the cell-free supernatants were collected after 72 h of eluate exposition
to the cells and the quantication of interleukin 1 (IL1), interleukin 6
(IL6), tumor necrosis factor (TNF ) and, macrophage inammatory
protein 1 (CCL3/MIP1) were performed in triplicate (da Silva et al.,
2016; Oliveira and Santos, 2011; Trubiani et al., 2012) by using a total
volume of 100 L of the supernatant, according to the manufacturer's
recommendations.
The real time reverse transcription-polymerase chain reaction (RT-
PCR) was performed for quantitative analysis of gene expression for
type IV collagen (COL IV) (COL4A3BP: Hs00178621_m1), matrix metal-
loproteinase 9 (MMP9) (MMMP9: Hs00234579_m1) and transforming
growth factor (TGF ) (TGFB1: Hs0099133_m1) (da Silva et al., 2016;
Oliveira and Santos, 2011), except for the P group.
The P group was not evaluated in the present study, since the acrylic
pigment could be incorporated into the resin during the manufacture of
the ocular prosthesis, though its use does not occur in an isolated form.
182 E.V.F. da Silva et al. / Toxicology in Vitro 36 (2016) 180185

The total RNA extraction from the cells was performed by using
TRIzol reagent (Invitrogen Life Technologies, California, United States),
according to the manufacturer's instructions, after 72 h of eluate expo-
sition to the cells. The RNA concentration was measured through spec-
trophotometry and the rst strand cDNAs were synthesized using 1 g
of total RNA and Superscript II RNase H reverse transcriptase
(Invitrogen Life Technologies, California, United States). After this
stage, mRNA levels for COL IV, MMP9 and TGF were measured and
their amplication was performed by a StepOnePlus Real-Time PCR Sys-
tem (Applied Biosystems, Invitrogen Life Technologies, California, Unit-
ed States). The internal control was the mRNA for -actin (ACTB:
Hs03023880_g1). The assay was run in duplicate for each sample by Fig. 2. IL6 concentration for the evaluated materials. C: negative control group. R: acrylic
using a volume of 20 L. The thermal cycling conditions determined resin without pigment incorporation. RP: acrylic resin with pigment incorporation. P:
by the manufacturer were followed. The results were analyzed using acrylic pigment. The results show mean standard error of IL6 concentration (pg/mL).
Different capital letters indicate statistical difference (p b 0.05) between groups.
the comparative threshold cycle (CT) (da Silva et al., 2016).

2.1. Statistical analysis

The distribution of each measurement was analyzed for the assump-

tion of normality. Quantitative data were normally distributed. There-
fore, one-factor analysis of variance (ANOVA) with Bonferroni post-
tests (p b 0.05) was conducted, using SPSS 21.0 statistics software
(IBM Incorporation, New York, United States). The percentage of chem-
ical elements present on the sample surface was compared among
groups.

3. Results
The percentage of cell proliferation for the materials with different
pigmentations evaluated in this study is shown in Fig. 1. Statistical dif-
ference was observed between groups (p = 0.000). The R (88.4%) and
P (87.5%) groups presented lower percentages of cell proliferation,
with statistical difference from the C group (100%).
No detectable levels of IL1 and CCL3/MIP1 were observed in the
present study. On the other hand, high levels of IL6 were found for the
tested groups (Fig. 2) and signicantly varied according to the material
evaluated (p = 0.000). Higher concentrations were observed for the RP
group (14.708 pg/mL), when compared to the C (9.842 pg/mL) and P
(3.312 pg/mL) groups. No statistical difference was veried in the levels
of IL6 between the R group and the C and RP groups.
Fig. 3 Illustrates the absence of statistically signicant difference in
the levels of TNF for the materials with different pigmentations eval-
uated (p = 0.264).
Through Fig. 4, which shows the relative quantication of mRNA for
COL IV for the materials tested, a statistical difference was veried be-
tween groups (p = 0.000). The R (2.21) and C (2.01) groups exhibited
higher gene expression of COL IV than the RP group (1.25), with statis-
tical difference.
Fig. 3. TNF concentration for the evaluated materials. C: negative control group. R:
acrylic resin without pigment incorporation. RP: acrylic resin with pigment
incorporation. P: acrylic pigment. The results show mean standard error of TNF
concentration (pg/mL). Different capital letters indicate statistical difference (p b 0.05)
between groups.
Based on Fig. 5, a statistical difference in the relative quantication of
mRNA for MMP9 was observed between groups (p = 0.03). The R
(5.75) and C (5.77) groups expressed more genes of MMP9 than the
RP group (2.95), with statistically signicant difference.
Through Fig. 6, it is possible to observe the relative quantication of
mRNA for TGF for the materials tested. There was a statistical differ-
ence between the groups (p = 0.000), wherein the R group (2.02)
expressed more genes of TGF , when compared to the RP group (1.34).
The chemical elements present on the sample surface were identi-
ed by EDS test (Table 2). The elements carbon (C) and oxygen (O)
were detected in all groups. The RP group had, in addition, 2.14% silicon
(Si). In the P group, 1.00% Si and 0.13% aluminum (Al) were found.

Fig. 1. Percentage of cell proliferation for the evaluated materials. C: negative control
group. R: acrylic resin without pigment incorporation. RP: acrylic resin with pigment
incorporation. P: acrylic pigment. T: Tween. The results show mean standard error of
cell proliferation percentage. Different capital letters indicate statistical difference
(p b 0.05) compared to the respective group C.
Fig. 4. Relative quantication of mRNA for COL IV for the evaluated materials. C: negative
control group. R: acrylic resin without pigment incorporation. RP: acrylic resin with
pigment incorporation. P: acrylic pigment. The results show mean standard error of
mRNA expression for COL IV. Different capital letters indicate statistical difference
(p b 0.05) between groups.
 E.V.F. da Silva et al. / Toxicology in Vitro 36 (2016) 180185
Fig. 5. Relative quantication of mRNA for MMP9 for the evaluated materials. C: negative
control group. R: acrylic resin without pigment incorporation. RP: acrylic resin with
pigment incorporation. P: acrylic pigment. The results show mean standard error of
mRNA expression for MMP9. Different capital letters indicate statistical difference
(p b 0.05) between groups.
4. Discussion
The null hypothesis that the N1 color acrylic resin, with or without
acrylic pigment, and the isolated acrylic pigment do not produce toxic
effects on the cell line studied was not accepted, since the materials
with different pigmentations showed divergent behaviors regarding
the performed tests.
In the present study, it was observed that all groups had a cell prolif-
eration higher than 75% (Fig. 1), suggesting that the materials are con-
sidered non-cytotoxic, according to ISO 10993-5 standard, which
evaluates the in vitro methods for the analysis of cytotoxicity
(International Organization for Standardization [homepage], 2009).
These results are consistent with the ndings from Retamoso et al.
(2014), that found that acrylic pigments used in chemically activated
resins did not inuence the biocompatibility of the material.
Although not cytotoxic, the P group had the lowest percentage of cell
proliferation (Fig. 1). This may be associated with the presence of differ-
ent chemical elements in the composition of the material. Through EDS
analysis, the elements aluminum and silicon were detected on the sur-
face of the sample of this group (Table 2).
It is known that aluminum is one of the most abundant elements on
the earth's crust in the form of aluminum oxide (Al2O3) (World Health
Organization [homepage], 2010). According to the World Health Orga-
nization, currently, the tolerable weekly dose is 1 mg of aluminum per
kilogram of body weight (World Health Organization [homepage],
2010). Studies have shown that the immune system seems to be sensi-
tive to exposure to Al (World Health Organization [homepage], 2010;
Fig. 6. Relative quantication of mRNA for TGF for the evaluated materials. C: negative
control group. R: acrylic resin without pigment incorporation. RP: acrylic resin with
pigment incorporation. P: acrylic pigment. The results show mean standard error of
mRNA expression for TGF . Different capital letters indicate statistical difference
(p b 0.05) between groups.
183
Table 2
Percentage (%) of each chemical compound for the groups assessed by energy-dispersive
spectroscopy (EDS).
Groups
Chemical compound R RP P
Carbon 75.79% 74.45% 75.02%
Oxygen 24.21% 23.41% 23.85%
Silicon - 2.14% 1.00%
Aluminum - - 0.13%
R: acrylic resin without pigment incorporation. RP: acrylic resin with pigment incorpora-
tion. P: acrylic pigment.
Zhu et al., 2014; Zuh et al., 2013). Some people manifest allergy to alu-
minum, suffering from dermatitis to its contact, including digestive dis-
orders by eating food cooked in aluminum recipients (World Health
Organization [homepage], 2010; Zhu et al., 2014; Zuh et al., 2013). Alu-
minum is not considered as toxic as heavy metals, but there is some ev-
idence of a certain toxicity when ingested in large quantities,
particularly when in the form of ions, because it is soluble in water
(World Health Organization [homepage], 2010; Zhu et al., 2014; Zuh
et al., 2013). Although the P group was not cytotoxic, the working hy-
pothesis is that cell proliferation may have been reduced due to the
presence of aluminum ions.
A statistically signicant larger concentration of IL6 for the RP group,
when compared to the C group, was veried (Fig. 2). According to EDS
assay, the samples of acrylic resin with acrylic pigment (group RP) pre-
sented, besides C and O, the element Si (Table 2).
The presence of toxic concentrations of metal ions, including silicon,
can be associated with genetic alterations, cytotoxicity, inammatory
and carcinogenic reactions (Sargeant and Goswami, 2007). According
to Speck-Hernandez and Montoya-Ortiz (2012), the cytotoxic process
resulting from the exposure to materials containing silicon include the
reduction of cell metabolic activity, the increase of production of inam-
matory mediators, such as proinammatory cytokines (IL1, IL6 and
TNF ) and apoptosis. The working hypothesis is that a similar reaction
may have occurred in this study, due to the release of silicon ions in the
culture medium and their subsequent exposure to the cell culture.
Detectable concentrations of IL1 and CCL3/MIP1 were not found,
suggesting that the stimulus given by the eluates of the tested materials
was not sufcient for the Chang conjunctival cells to secrete such in-
ammatory mediators.
Although no statistically signicant difference was found in concen-
trations of TNF , numerically larger quantities of this mediator can be
observed for the evaluated materials when compared to the C group
(Fig. 3). TNF is one of the main pro-inammatory cytokines and has,
together with IL1 and IL6, the ability to increase the local concentration
of cells for tissue repair. One of the functions of this mediator is to pro-
mote immune and inammatory responses by recruiting neutrophils
and monocytes to the infection site, and activate them (Abbas et al.,
2011).
Regarding the relative quantication of mRNA for COL IV, MMP9 and
TGF , larger amounts of these targets have occurred for the C and R
groups (Figs. 4 to 6), with no statistically signicant difference between
them, indicating that the Chang conjunctival cell line seems to produce
these targets in physiological condition. Similarity was observed in the
increase of gene expression of COL IV and TGF (protein responsible
for stimulating COL IV synthesis) (Abbas et al., 2011; Romi et al.,
2012). It is known that the balance between the breakage and the syn-
thesis of collagen is essential, while the tissue repair occurs, aiming to
avoid brous reaction (Tirado-Rodriguez et al., 2014; Santibaez et al.,
2011). The COL IV composes the basement membrane extracellular ma-
trix of epithelial cells. It is crucial for a balance to exist in mRNA expres-
sion of COL IV, MMP9 and TGF , for the preservation of cell structure.
However, the expression of MMP9, that acts on the degradation of
COL IV (Abbas et al., 2011; Romi et al., 2012), was about three times
higher than the expression of the other targets, which can be
184 E.V.F. da Silva et al. / Toxicology in Vitro 36 (2016) 180185
detrimental, since the excessive activation of MMP9 damages cell mor-
phology (Yang et al., 2007).
It is also important to point out that there are basically two tech-
niques of articial pigmentation of the sclera to try to more naturally
simulate the ocular prosthesis, the extrinsic and intrinsic techniques,
which may or may not be associated. The extrinsic technique consists
of applying pigment on the external surface of the prosthesis base
after the acrylization, by using pigments solubilized in MMA monomer
that will later be covered by a thin layer of colorless resin. The intrinsic
technique is based on the incorporation of small layers of pigment in the
acrylic resin mass that will be subsequently polymerized (Goiato et al.,
2013). In this study, the intrinsic technique was used and the results ob-
tained did not affect cell proliferation. However, it is necessary to exe-
cute further studies to verify the other technique, which may be
related or unrelated to the increased release of subproducts, which
may cause toxic effects on the cells. Additionally, studies to verify and
quantify ions that can be released after polymerization of the studied
materials should be performed.
In summary, the materials exhibited divergent biological behavior,
regarding cytotoxicity, cell activation and production of matrix proteins.
The RP group showed statistically similar results to the C group, regard-
ing cell proliferation and was not cytotoxic, despite increasing the pro-
duction of IL6.
Conicts of interest statement
None declared.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgments
The authors thank CNPq (National Council for Scientic and Techno-
logical Development), CAPES (Coordination for the Improvement of
Higher Education Personnel) and FAPESP (Foundation for Support to
Research of the State of Sao Paulo) (# 2013/11830-4) for the scholarship
granted to the rst author.
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